Raman spectroscopy is a spectroscopic technique used in condensed matter physics, chemistry, biology and medical diagnostics, among others, to study vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. Typically, photons are absorbed or emitted by the laser light, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the phonon modes in the system. Infrared spectroscopy yields similar, but complementary information.
Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is collected with a lens and sent through a monochromator. Wavelengths close to the laser line (due to elastic Rayleigh scattering) are filtered out and those in a certain spectral window away from the laser line are dispersed onto a detector.
Spontaneous Raman scattering is typically very weak, and as a result the main difficulty of Raman spectroscopy is separating the weak inelastically scattered light from the intense Rayleigh scattered laser light. Raman spectrometers typically use holographic diffraction gratings and multiple dispersion stages to achieve a high degree of laser rejection. A photon-counting photomultiplier tube (PMT) or, more commonly, a CCD camera is used to detect the Raman scattered light.
The Raman effect occurs when light impinges upon a molecule and interacts with the electron cloud of the bonds of that molecule. The amount of deformation of the electron cloud is the polarizability of the molecule. The amount of the polarizability of the bond will determine the intensity and frequency of the Raman shift. The photon (light quantum), excites one of the electrons into a virtual state. When the photon is released the molecule relaxes back into a vibrational energy state as shown in FIG. 1. For example, when the molecule relaxes into the zero vibrational energy state (i.e., “ground state”), it generates Rayleigh scattering. The molecule could relax into the first vibration energy states, and this generates Stokes Raman scattering. However, if the molecule was already in an elevated vibrational energy state such as the first vibrational energy state and it relaxes into the zero vibrational energy state, the Raman scattering is then called Anti-Stokes Raman scattering. By Stokes Raman scattering, the wavelength of the emitted light is longer than the wavelength of the excitatory light. By anti-Stokes Raman scattering, the wavelength of the emitted light is shorter that the wavelength of the excitatory light.
The sensitive and accurate detection, identification and multiplexed molecular imaging of different chemical/biological composition inside a sample with single molecule sensitivity and high multiplicity has not been done. Even the detection and identification of small numbers (<1000) of molecules from biological and other samples has proven to be an elusive goal, despite widespread potential uses in medical diagnostics, pathology, toxicology, environmental sampling, chemical analysis, forensics and numerous other fields. The embodiments of this invention address these problems in the current state of the art.